A High-Content Analysis Toolbox Permits Dissection of Diverse Signaling Pathways for T Lymphocyte Polarization

The Journal of Immunology

O’Dowd

Paul

et al. 2012

Self Cite

105

Chemokines such as SDF-1α play a crucial role in orchestrating T lymphocyte polarity and migration via polymerization and reorganization of the F-actin cytoskeleton, but the role of actin-associated proteins in this process is not well characterized. In this study, we have investigated a role for L-plastin, a leukocyte-specific F-actin–bundling protein, in SDF-1α–stimulated human T lymphocyte polarization and migration. We found that L-plastin colocalized with F-actin at the leading edge of SDF-1α–stimulated T lymphocytes and was also phosphorylated at Ser5, a site that when phosphorylated regulates the ability of L-plastin to bundle F-actin. L-plastin phosphorylation was sensitive to pharmacological inhibitors of protein kinase C (PKC), and several PKC isoforms colocalized with L-plastin at the leading edge of SDF-1α–stimulated lymphocytes. However, PKC ζ, an established regulator of cell polarity, was the only isoform that regulated L-plastin phosphorylation. Knockdown of L-plastin expression with small interfering RNAs demonstrated that this protein regulated the localization of F-actin at the leading edge of chemokine-stimulated cells and was also required for polarization, lamellipodia formation, and chemotaxis. Knockdown of L-plastin expression also impaired the Rac1 activation cycle and Akt phosphorylation in response to SDF-1α stimulation. Furthermore, L-plastin also regulated SDF-1α–mediated lymphocyte migration on the integrin ligand ICAM-1 by influencing velocity and persistence, but in a manner that was independent of LFA-1 integrin activation or adhesion. This study, therefore, demonstrates an important role for L-plastin and the signaling pathways that regulate its phosphorylation in response to chemokines and adds L-plastin to a growing list of proteins implicated in T lymphocyte polarity and migration.

Section: Resultsmentioning

confidence: 99%

L-Plastin Regulates Polarization and Migration in Chemokine-Stimulated Human T Lymphocytes

The Journal of Immunology

O’Dowd

Paul

et al. 2012

Self Cite

105

“…High Content Analysis-A high content analysis protocol for T-cell morphology analysis has been optimized and established in our laboratory as described (29,34). Briefly, cells were seeded in triplicate on 96-well flat bottom plates precoated with either poly-L-lysine or anti-LFA-1 for 2 h. After washing, cells were fixed by incubating them for 20 min with 3% (w/v) paraformaldehyde in PBS.…”

Section: Methodsmentioning

confidence: 99%

“…We employed objective quantification of cytoskeletal change using a previously reported cell-based method of high content analysis that entailed population analysis of the morphology of cells expressing GFP fusion protein (34). Utilizing this approach, we observed that although cells expressing the Rab5a-T7A-GFP mutant were polarized after anti-LFA-1 stimulation, the cell polarization index based on the actin cytoskeleton was significantly reduced (p ϭ 0.003) as compared with cells expressing wild-type Rab5a-GFP or Rab5a-T7E-GFP (Fig.…”

Section: T7a Mutation Partially Inhibits Rab5amentioning

confidence: 99%

Phosphorylation of Rab5a Protein by Protein Kinase Cϵ Is Crucial for T-cell Migration

Ong

Journal of Biological Chemistry

Skubis-Zegadło

et al. 2014

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Background: Rab5a GTPase plays important roles in intracellular transport and cell signaling. Results: T-cell stimulation through the integrin LFA-1 or the chemokine receptor CXCR4 induces PKC⑀-dependent phosphorylation of Rab5a at Thr-7, which is crucial for cytoskeleton remodeling and cell migration. Conclusion: PKC⑀-Rab5a-Rac1 axis regulates T-cell motility. Significance: The study provides novel insights into the role of Rab5a in the adaptive immune response.

“…Involvement of a number of serine/threonine kinases including protein kinase C and myosin light chain kinase in T-cell migratory processes has previously been established (Volkov et al, 1998(Volkov et al, , 2001Smith et al, 2003). Of note, other tyrosine kinase inhibitors including Herbimycin A (a broad-spectrum inhibitor of non-receptor tyrosine kinase), piceatannol (a Syk-selective tyrosine kinase inhibitor) and a dominant negative ZAP-70 tyrosine kinase have been shown to significantly reduce LFA-1 triggered T-cell migratory processes by our research group (Freeley et al, 2010;Verma et al, 2009;Kelleher et al, 1995) and others (Soede et al, 1998;Wang et al, 2009). Collectively these findings suggest that early tyrosine phosphorylation of proteins is an important event in T-cell migration.…”

Section: Tyrosine Phosphorylation Of Proteins In T-cell During Lfa-1 mentioning

confidence: 99%

Analysis of dynamic tyrosine phosphoproteome in LFA‐1 triggered migrating t‐cells

Verma

Dempsey

Journal Cellular Physiology

et al. 2011

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The ordered, directional migration of T-lymphocytes is a key process during immune surveillance and response. This requires cell adhesion to the high endothelial venules or to the extracellular matrix by a series of surface receptor/ligand interactions involving adhesion molecules of the integrin family including lymphocyte function associated molecule-1 (LFA-1) and intercellular adhesion molecules (ICAMs). Reversible protein phosphorylation is emerging as a key player in the regulation of biological functions with tyrosine phosphorylation playing a crucial role in signal transduction. Thus, the study of this type of post-translational modification at the proteomic level has great biological significance. In this work, phospho-enriched cell lysates from LFA-1-triggered migrating human T-cells were subjected to immunoaffinity purification of tyrosine phosphorylated proteins, mass spectrometric and bioinformatic analysis.In addition to the identification of several well-documented proteins, the analysis suggested involvement of a number of new and novel proteins in LFA-1 induced T-cell migration. This dataset expands the list of the signalling components of the LFA-1 induced phosphotyrosine protein complexes in migrating T-cells that will be extremely useful in the study of their specific roles within LFA-1 associated signalling pathways. Identification of proteins previously not reported in the context of LFA-1 stimulated signal transduction may provide new insights into understanding the LFA-1 signalling networks and aid in the search for new potential therapeutic targets.